Enterobacteriaceae Clinical Bacteriology II CLS 413 Lab 1 CLS Department College of Applied Medical Sciences King Saud University Basmah Almaarik

Course Content Enterobacteriacae Other gram –ve bacteria Lactose Fermenters: Escherichia coli Klebsiella Pneumoniae Lactose Non Fermenters Salmonella Shigella Proteus Other gram –ve bacteria Pseudomonas, Hemophilus, Vibrio, Campylobacter Spirochetes Anaerobes Clostridia and Bacteroides The genus Pseudomonas belongs to the family Pseudomonadacea The Genus hemophilus belongs to family Pasteurellaceae

Introduction Enterobacteriaciae are a large family of Gram-negative rod-shaped bacteria that includes, along with many harmless normal flora, many of the medically significant pathogens General Characteristics of Enterobacteriacea: Rod shaped Gram negative Catalase positive Oxidase negative Facultative anaerobes Most ferment glucose, some are LF Most reduce nitrate to nitrite Gram Negative Bacilli of Escherichia coli from a woman suffering from Urinary tract infection

Members of the Enterobacteriaceae Family  E.coli:  lactose fermenter- releases acids as a product of fermentation. Color (Ph) indicators change the color of the medium in response to the acidic environment.  Usually produces dry LF colonies  Motile  Klebsiellah Pneumoniae:  Lactose fermenter  Mucoid LF colonies Isolated brick-red coloured E. coli colonies surrounded by a precipitated zone on MAC agar K.pneumoniae growing on MacConkey

Members of the Enterobacteriaceae Family  Proteus:  Non lactose fermenter (NLF)  Tends to swarm on most culture media  CLED agar prevents swarming  Can produce H2S in sodium thiosulfate-containing media  Motile  Salmonella:  NLF  Produces H2S in sodium thiosulfate-containing media  motile  Shigella:  Non H2S producer

Desoxycholate Citrate Agar (DCA): Differential and selective. Selective for gram –ve Inhibitory substances: sodium desoxycholate and sodium citrate Differentiates between LF and NLF and between H2S producers and non producers Neutral red is pH indicator H2S producers give black-centered colorless colonies on DCA (salmonella). Shigella sp. produce colorless colonies without a black center. LFs give pink colonies, while lactose NLFs produce colorless or pale colonies

H2S-producers on DCA

Shigella (non H2S producer) on DCA Indicator color chart:

Hektoen Enteric Agar (HE)  Differential and Selective  Selective for gram –ve bacteria (Contains bile salt inhibitors)  Differentiation based on lactose fermentation and H2S production  Carbohydrate sources are lactose sucrose and salicin  Bromothymol blue is color indicator  Determines H2S production through sodium thiosulphate(as an H2S source)  H2S producers give clear colonies with black centers (fish eye colonies)  Lactose fermenters give orange colored colonies (sometimes called salmon colonies). Non fermenters give green-blue colonies

Hektoen Enteric Agar (HE) E.Coli salmon red colonies on HE Salmonella colonies with black center Lac –ve, H2S -ve colonies on HE Characteristic Fish- eye Colonies

Xylose Lysine Desoxycholate Agar (XLD):  A Selective and differential medium  Sodium desoxycholate is inhibitor  Phenol red: pH indicator  Differentiation: LF, H2S production  Contains xylose ,lactose (sugars) and lysine (amino acid)  LFs produce yellow colonies  H2s producers (salmonella): red colonies with black centers.  Non H2S produces red colonies without black centers  **All enterics, except shigella, ferment xylose this explains why proteus –even though it’s NLF- gives yellow colonies on XLD  Salmonella ferments xylose but, does not give yellow colonies  Why? because salmonella decarboxylates lysine in the medium leading to alkaline products (if the environment is alkaline, the plate remains red)

Salmonella on XLD: Lac –, H2S + red colonies with black centers Xylose Lysine Desoxycholate Agar (XLD): Lac +ve, H2S - colonies of on XLD Salmonella on XLD: Lac –, H2S + red colonies with black centers Shigella on XLD: lac -ve, H2S –ve: red colonies w/out black centers

How To Report a Gram Stain When reporting a gram stain, you need to describe 2 things: Whether the cells are gram positive or negative The shape and arrangement of the cells Shapes can be (bacilli, cocci, coccobacilli, spirohetes …etc) Arrangements can be (in chains, in clusters, in pairs (diplococci)..etc) NEVER mention the name of an organisms when you are asked to comment on a gram stain under the microscope. The gram stain by itself is insufficient to identify an organism. Identification can only be made after doing cultures and biochemical tests

Tasks to be done today 1- Prepare a smear stain it by the gram stain method. View the slide under the microscope. 2- Culture the given organisms on your agar plates. Then incubate at 37 degrees Celsius. Return to the lab within 24 hrs to record your results. Growth patterns may change after that and your plate reading will be inaccurate. 3- View the demonstration plates and prepare a table of growth characteristics (which you will include in your report). 4- Prepare your lab report as described before Make sure to include pictures of your results in your reports

Describe: Growth Color Lac fermentation • H2S +/- Specfic characteristics (mucoid colonies, dry, swarming..etc)

Reports Should Include Your name Practical Title Principle of the test performed Organism used for experiment Materials and reagents used (include not just gram stain materials, but also the materials you used to make the bacterial smear) Procedures (must include procedure of both bacterial smear preparation and gram stain) Result

Before you leave: Turn off your microscopes Turn off the incinerators Clean your bench with: disinfectant spray and tissue Wash your hands Return lab chair to place